Pharmaceutical formulation

ABSTRACT

A pharmaceutical formulation for a PKC modulatory peptide and a transport moiety comprising the aforementioned components and an anti-aggregant.

RELATED APPLICATION

This application claims benefit of U.S. application Ser. No. 60/615,486filed Sep. 30, 2004 which is hereby incorporated by reference in itsentirety.

TECHNICAL FIELD

This invention relates to pharmaceutical formulations, particularly toformulations of amino acids, peptides and small proteins, andspecifically to formulations for PKC peptide/transporter conjugates.

BACKGROUND ART

Protein kinase C (“PKC”) is a key enzyme in signal transduction involvedin a variety of cellular functions, including cell growth, regulation ofgene expression, and ion channel activity. The PKC family of isozymesincludes at least 10 different protein kinases that can be divided intoat least three subfamilies based on their homology and sensitivity toactivators. (See FIG. 1.) Each isozyme includes a number of homologous(“conserved” or “C”) domains interspersed with isozyme-unique(“variable” or “V”) domains. Members of the “classical” subfamily, α(SEQ ID NO:164), β_(I) (SEQ ID NO:165), β_(II) (SEQ ID NO:166) and γPKC(SEQ ID NO:167), contain four homologous domains (C1, C2, C3 and C4) andrequire calcium, phosphatidylserine, and diacylglycerol or phorbolesters for activation. Members of the “novel” subfamily, δ(SEQ IDNO:168), ε (SEQ ID NO:169), η (SEQ ID NO:170) and θPKC (SEQ ID NO:171),lack the C2 homologous domain and do not require calcium for activation.Finally, members of the “atypical” subfamily, ζ (SEQ ID NO:174) andλ/lPKC (SEQ ID NO:172), lack both the C2 and one-half of the C1homologous domains and are insensitive to diacylglycerol, phorbol estersand calcium.

Individual isozymes of PKC have been implicated in the mechanisms ofvarious disease states, including the following: cancer (alpha and deltaPKC); cardiac hypertrophy and heart failure (beta I and beta II PKC)nociception (gamma and epsilon PKC); ischemia including myocardialinfarction (delta and epsilon PKC); immune response, particularly T-cellmediated (theta PKC); and fibroblast growth and memory (zeta PKC).

DISCLOSURE OF THE INVENTION

In accordance with the objects outlined above, the disclosed inventionprovides a pharmaceutical formulation for a protein kinase C modulatorypeptide and a cationic (i.e., positively charged) transport peptide andan anti-aggregant. A preferred anti-aggregant is a sugar characterizedby having a sufficient number stereochemically aligned hydroxyl moietiesto interact with the modulatory peptide and/or the transport peptidehydrophobic and/or positively charged portions so as to favor theirorganization with the anti-aggregant, rather than aggregation with eachother. PKC modulatory peptides, such as peptides derived from variousPKC variable regions, comprise preferred embodiments. Cationic transportmoieties useful in the invention include cationic peptides, such aspoly-arginine and HIV-tat. A particularly preferred embodiment comprisesa PKC inhibitory peptide and a HIV-tat derived transport peptide. Anexample of such an embodiment is KAI-9803 (SEQ ID NO:1).

In one of the particular aspects of the above-described pharmaceuticalformulation, the ratio of anti-aggregant to peptide/transporterconjugate ranges from about 100:1 to about 1:1, 90:1, 80:1, 70:1, 60:1,50:1, 40:1, 30:1, 20:1, 10:1, 5:1, and 1:1.

Another aspect of the invention provides a stable pharmaceutical productfor shipping and storing prior to use, including a lyophilized cake ofKAI-9803 and an anti-aggregant in a sealed container. The lyophilizedproduct is preferably obtained from a solution of KAI-9803 plus acetatecounterion. The ratio of KAI-9803 to anti-aggregant is from about 1:5 toabout 1:100, particularly about 1:80 and especially about 1:8. Theanti-aggregant is preferably a sugar. One specific such product is 5 mgKAI-9803 and 40 mg mannitol in a stoppered glass vial. Instructions forreconstitution are preferably incorporated on the container or itsattached label, outer packaging and/or package insert.

Another aspect of the invention provides a formulation for parenteral(particularly intracoronary) administration that is about 2.5 mg/mLKAI-9803 and about 20 mg/mL mannitol reconstituted from a lyophilizedcake using sodium chloride for injection, USP (preferably 0.9%) to aconcentration ranging from about 0.001 to 2.5 mg/mL, preferably about0.01 to 1.0 mg/mL. To reconstitute the lyophilized formulation foradministration, a sealed container of product is first warmed to aboutroom temperature, after which a pharmaceutically acceptable solvent(such as saline, preferably 9% saline) is added in an amount sufficientto solubilize the lyophilized cake, followed by the addition of suchadditional quantity of solvent as is necessary to obtain a desiredconcentration for administration.

Still another aspect of the invention is a method of manufacture,including the steps:

-   (a) Appropriate amounts of anti-aggregant, hydrophobic active agent    and/or cationic transport moiety are introduced to a suitably sized    container (preferably a glass vial) as dry solids.-   (B) A pharmaceutically acceptable solvent is added to the container    in an amount sufficient to dissolve the solids.-   (C) The solution thus-formed is lyophilized to dryness.-   (D) The container is sealed (optionally after first filling the    head-space with a non-reactive gas, such as nitrogen).

Other aspects and embodiments will be apparent to those skilled in theart form the following detailed description.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows a schematic of the three families of protein kinase Cisozymes.

MODES OF CARRYING OUT THE INVENTION

The presently described invention relates to pharmaceutical formulationsof peptides which modulate the activity of one or more protein kinase Cisozymes. In certain embodiments, the peptides discussed herein arecoupled to a carrier moiety to facilitate transport of the modulatorypeptide to a target cell. Typically, preferred embodiments of thedisclosed pharmaceutical formulations further comprise an anti-aggregantand one or more excipients. The pharmaceutical formulations comprisingthe modulatory peptides provide advantages in the handling of the activepharmaceutical ingredients, in formulation manufacture, stability,concentration and ease of use. These and other advantages are describedin greater detail below.

DEFINITIONS

As used in the present, specification, the following words and phrasesare generally intended to have the meanings as set forth below, exceptto the extent that the context in which they are used indicatesotherwise.

A “PKC modulatory peptide” or “a peptide which modulate the activity ofone or more protein kinase C isozymes” refer to a peptide that canpromote, enhance or activate one or more PKC isozymes, or alternativelythe peptide can also inhibit or inactivate one or more PKC isozymes.

The term “API” means active pharmaceutical ingredient, which as usedherein refers to a PKC modulatory peptide and a transport moiety,covalently bound to one another, and/or one or more active agents.

The term “disorder” or “disease state” means any mammalian disease,condition, symptom, or indication, preferably arising in a humanpatient.

The term “effective amount” refers to that amount of an API that issufficient to effect treatment, as defined below, when administered to amammal in need of such treatment.

The term “KAI-9803” refers to an peptide derived from the first variableregion of δPKC conjugated via a Cys-Cys disulfide linkage to a HIVTat-derived transporter peptide, and can be represented as follows:

The term “optional” or “optionally” means that the subsequentlydescribed event or circumstance may or may not occur, and that thedescription includes instances where such event or circumstance occursand instances in which it does not.

As used herein, “pharmaceutically acceptable carrier” or“pharmaceutically acceptable excipient” includes any and all solvents,dispersion media, antibacterial and antifungal agents, isotonic andabsorption delaying agents and the like. The use of such media andagents for pharmaceutically active substances is well known in the art.Supplementary active ingredients can also be incorporated into thecompositions.

The term “pharmaceutically acceptable salt” or “counterion” refers tosalts which retain the biological effectiveness and properties of theAPI and which are not biologically or otherwise undesirable. In manycases, the API will be capable of forming acid and/or base salts byvirtue of the presence of amino and/or carboxyl groups or groups similarthereto. Pharmaceutically acceptable base addition salts can be preparedfrom inorganic and/or organic bases. Pharmaceutically acceptable acidaddition salts may be prepared from inorganic and/or organic acids. Forexample, inorganic acids include hydrochloric acid, hydrobromic acid,sulfuric acid, nitric acid, phosphoric acid, and the like. Organic acidsinclude acetic acid, propionic acid, glycolic acid, pyruvic acid, oxalicacid, malic acid, malonic acid, succinic acid, maleic acid, fumaricacid, tartaric acid, citric acid, benzoic acid, cinnamic acid, mandelicacid, methanesulfonic acid, ethanesulfonic acid, p-toluene-sulfonicacid, salicylic acid, and the like.

The term “pharmaceutical product” refers to an API, formulated andfilled into a container for storage, transportation, or administration.

The term “PKC-derived peptide” refers to a PKC isozyme- and/or variableregion-specific peptides as described, for example, in U.S. Patents andPublications U.S. Pat. Nos. 5,783,405, 6,165,977, US2002/0150984,US2002/0168354, US2002/057413, US2003/0223981, US2004/0009922 and incopending U.S. provisional application Ser. No. 60/550,755, filed Mar.5, 2004, all of which are hereby incorporated by reference in theirentirety.

The term “transporter moiety” means a component of an API thatfacilitates cellular uptake, such as cationic polymers, peptides andantibody sequences, including polylysine, polyarginine,Antennapedia-derived peptides, HIV Tat-derived peptides and the like. Anexample of a transporter moiety is a “transporter peptide”, which is apeptide which facilitates cellular uptake of a PKC modulating peptidewhich is chemically associated or bonded to the transporter peptide.

The term “treatment” or “treating” means any treatment of a disease ordisorder in a mammal, including: preventing or protecting against thedisease or disorder, that is, causing the clinical symptoms not todevelop; inhibiting the disease or disorder, that is, arresting orsuppressing the development of clinical symptoms; and/or relieving thedisease or disorder, that is, causing the regression of clinicalsymptoms.

The term “prophylaxis” is intended as an element of “treatment” toencompass both “preventing” and “suppressing” as defined herein. It willbe understood by those skilled in the art that in human medicine it isnot always possible to distinguish between “preventing” and“suppressing” since the ultimate inductive event or events may beunknown, latent, or the patient is not ascertained until well after theoccurrence of the event or events.

Protein Kinase C Modulatory Peptides

Various PKC isozyme- and variable region-specific peptides have beendescribed and can be used with the presently disclosed invention.Preferably, the PKC modulatory peptide is a V1, V3 or V5-derivedpeptide. The following US Patents or Patent Applications describe avariety of suitable peptides that can be used with the presentlydisclosed invention: U.S. Pat. Nos. 5,783,405, 6,165,977, 6,855,693,US2004/0204364, US2002/0150984, US2002/0168354, US2002/057413,US2003/0223981, US2004/0009922 and Ser. No. 10/428,280, each of whichare incorporated herein by reference in their entirety. Table 1 providesa listing of preferred PKC modulatory peptides for use with the presentinvention.

TABLE 1 Peptides derived from PKC isozymes Peptide SEQ ID NO. SequenceαV3-1 SEQ ID NO:2 I-P-E-G-D-E-E-G αV5-1 SEQ ID NO:3 Q-L-V-I-A-N αV5-1.1SEQ ID NO:4 G-L-G-A-E-N αV5-1.2 SEQ ID NO:5 A-R-G-A-E-N αV5-1.3 SEQ IDNO:6 C-G-K-G-A-E-N αV5-1.4 SEQ ID NO:7 C-G-K-G-A-E-N βC2-1 SEQ ID NO:8K-Q-K-T-K-T-I-K βC2-2 SEQ ID NO:9 M-D-P-N-G-L-S-D-P-Y-V-K-L βC2-3 SEQ IDNO:10 I-P-D-P-K-S-E βC2-4 SEQ ID NO:11 S-L-N-P-E-W-N-E-T βV3-1 SEQ IDNO:12 V-P-P-E-G-S-E-A βIV5-1 SEQ ID NO:13 K-L-F-I-M-N βIV5-2 SEQ IDNO:14 R-D-K-R-D-T-S βIV5-2.1 SEQ ID NO:15 C-A-R-D-K-R-D-T-S βIV5-2.2 SEQID NO:16 G-R-D-K-R-D-T-S βIV5-2.3 SEQ ID NO:17 A-R-D-K-R-D-T-S βIV5-3SEQ ID NO:18 A-R-D-K-R-D-T-S-N-F-D-K βIV5-4 SEQ ID NO:19A-G-F-S-Y-T-N-P-E-F-V-I-N-V βIIV5-1 SEQ ID NO:20 Q-E-V-I-R-N βIIV5-2 SEQID NO:21 C-G-R-N-A-E βIIV5-3 SEQ ID NO:22 A-C-G-R-N-A-E βIIV5-3.1 SEQ IDNO:23 A-C-G-K-N-A-E βIIV5-4 SEQ ID NO:24 K-A-C-G-R-N-A-E βIIV5-5 SEQ IDNO:25 C-G-R-N-A-E-N βIIV5-6 SEQ ID NO:26 A-C-G-R-N-A-E βIIV5-7 SEQ IDNO:27 S-F-V-N-S-E-F-L-K-P-E-V-L-S γV3-1 SEQ ID NO:28 V-A-D-A-D-N-C-SγV5-1 SEQ ID NO:29 G-R-S-G-E-N γV5-1.1 SEQ ID NO:30 G-L-S-G-E-N γV5-2SEQ ID NO:31 R-L-V-L-A-S γV5-3 SEQ ID NO:32 P-C-G-R-S-G-E-N δV1-1 SEQ IDNO:33 C-S-F-N-S-Y-E-L-G-S-L δV1-1.1 SEQ ID NO:34 S-F-N-S-Y-E-L-G-S-LδV1-1.2 SEQ ID NO:35 T-F-N-S-Y-E-L-G-S-L δV1-1.3 SEQ ID NO:36A-F-N-S-N-Y-E-L-G-S-L δV1-1.4 SEQ ID NO:37 S-F-N-S-Y-E-L-G-T-L δV1-1.5SEQ ID NO:38 S-T-N-S-Y-E-L-G-S-L δV1-1.6 SEQ ID NO:39S-F-N-S-F-E-L-G-S-L δV1-1.7 SEQ ID NO:40 S-N-S-Y-D-L-G-S-L δV1-1.8 SEQID NO:41 S-F-N-S-Y-E-L-P-S-L δV1-1.9 SEQ ID NO:42 T-F-N-S-Y-E-L-G-T-LδV1-1.10 SEQ ID NO:43 S-F-N-S-Y-E-I-G-S-V δV1-1.11 SEQ ID NO:44S-F-N-S-Y-E-V-G-S-I δV1-1.12 SEQ ID NO:45 S-F-N-S-Y-E-L-G-S-V δV1-1.13SEQ ID NO:46 S-F-N-S-Y-E-L-G-S-I δV1-1.14 SEQ ID NO:47S-F-N-S-Y-E-I-G-S-L δV1-1.15 SEQ ID NO:48 S-F-N-S-Y-E-V-G-S-L δV1-1.16SEQ ID NO:49 A-F-N-S-Y-E-L-G-S-L δV1-1.17 SEQ ID NO:50 Y-D-L-G-S-LδV1-1.18 SEQ ID NO:51 F-D-L-G-S-L δV1-1.19 SEQ ID NO:52 Y-D-I-G-S-LδV1-1.20 SEQ ID NO:53 Y-D-V-G-S-L δV1-1.21 SEQ ID NO:54 Y-D-L-P-S-LδV1-1.22 SEQ ID NO:55 Y-D-L-G-L-L δV1-1.23 SEQ ID NO:56 Y-D-L-G-S-IδV1-1.24 SEQ ID NO:57 Y-D-L-G-S-V δV1-1.25 SEQ ID NO:58 I-G-S-L δV1-1.26SEQ ID NO:59 V-G-S-L δV1-1.27 SEQ ID NO:60 L-P-S-L δV1-1.28 SEQ ID NO:61L-G-L-L δV1-1.29 SEQ ID NO:62 L-G-S-I δV1-1.30 SEQ ID NO:63 L-G-S-VδV1-2 SEQ ID NO:64 A-L-S-T-E-R-G-K-T-L-V δV1-2.1 SEQ ID NO:65A-L-S-T-D-R-G-K-T-L-V δV1-2.2 SEQ ID NO:66 A-L-T-S-D-R-G-K-T-L-V δV1-2.3SEQ ID NO:67 A-L-T-T-D-R-G-K-S-L-V δV1-2.4 SEQ ID NO:68A-L-T-T-D-R-P-K-T-L-V δV1-2.5 SEQ ID NO:69 A-L-T-T-D-R-G-R-T-L-V δV1-2.6SEQ ID NO:70 A-L-T-T-D-K-G-K-T-L-V δV1-2.7 SEQ ID NO:71A-L-T-T-D-K-G-K-T-L-V δV1-3 SEQ ID NO:72 V-L-M-R-A-A-E-E-P-V δV1-4 SEQID NO:73 Q-S-M-R-S-E-D-E-A-K δV1-5 SEQ ID NO:163 A-F-N-S-Y-E-L-G-S δV3-1SEQ ID NO:74 Q-G-F-E-K-K-T-G-V δV3-2 SEQ ID NO:75 D-N-N-G-T-Y-G-K-IδV5-1 SEQ ID NO:76 K-N-L-I-D-S δV5-2 SEQ ID NO:77 V-K-S-P-R-D-Y-SδV5-2.1 SEQ ID NO:78 V-K-S-P-C-R-D-Y-S δV5-2.2 SEQ ID NO:79I-K-S-P-R-L-Y-S δV5-3 SEQ ID NO:80 K-N-L-I-D-S δV5-4 SEQ ID NO:81P-K-V-K-S-P-R-D-Y-S-N εV1-1 SEQ ID NO:82 N-G-L-L-K-I-K εV1-2 SEQ IDNO:83 E-A-V-S-L-K-P-T εV1-3 SEQ ID NO:84 L-A-V-F-H-D-A-P-I-G-Y εV1-4 SEQID NO:85 D-D-F-V-A-N-C-T-I εV1-5 SEQ ID NO:86 W-I-D-L-E-P-E-G-R-V εV1-6SEQ ID NO:87 H-A-V-G-P-R-P-Q-T-F εV1-7 SEQ ID NO:88 N-G-S-R-H-F-E-DεV1-7.1 SEQ ID NO:89 H-D-A-P-I-G-D-Y εV1-7.2 SEQ ID NO:90 H-D-A-P-I-GεV1-7.3 SEQ ID NO:91 H-D-A-A-I-G-Y-D εV1-7.4 SEQ ID NO:92H-D-A-P-I-P-Y-D εV1-7.5 SEQ ID NO:93 H-N-A-P-I-G-Y-D εV1-7.6 SEQ IDNO:94 H-A-A-P-I-G-Y-D εV1-7.7 SEQ ID NO:95 A-D-A-P-I-G-Y-D εV1-7.8 SEQID NO:96 H-D-A-P-A-G-Y-D εV1-7.9 SEQ ID NO:97 H-D-A-P-I-G-A-D εV1-7.10SEQ ID NO:98 H-D-A-P-I-A-Y-D εV1-7.11 SEQ ID NO:99 H-D-A-P-I-G-Y-A εV3-1SEQ ID NO:100 S-S-P-S-E-E-D-R-S εV3-2 SEQ ID NO:101 P-C-D-Q-E-I-K-EεV3-3 SEQ ID NO:102 E-N-N-I-R-K-A-L-S εV3-4 SEQ ID NO:103G-E-V-R-Q-G-Q-A εV5-1 SEQ ID NO:104 E-A-I-V-K-Q εV5-2 SEQ ID NO:105I-K-T-K-R-D-V εV5-2.1 SEQ ID NO:106 I-K-T-K-R-L-I εV5-3 SEQ ID NO:107C-E-A-I-V-K-Q εV5-4 SEQ ID NO:108 T-K-R-D-V-N-N-F-D-Q ζV1-1 SEQ IDNO:109 V-R-L-K-A-H-Y ζV1-2 SEQ ID NO:110 V-D-S-E-G-D ζV1-3 SEQ ID NO:111V-F-P-S-I-P-E-Q ζV3-1 SEQ ID NO:112 S-Q-E-P-P-V-D-D-K-N-E-D-A- D-L ζV3-2SEQ ID NO:113 I-K-D-D-S-E-D ζV3-3 SEQ ID NO:114 P-V-I-D-G-M-D-G-I ζV5-1SEQ ID NO:115 E-D-A-I-K-R ζV5-1.1 SEQ ID NO:116 E-D-A-I-R ζV5-2 SEQ IDNO:117 I-T-D-D-Y-G-L-D ζV5-2.1 SEQ ID NO:118 I-T-D-D-Y-G-D-L ζV5-3 SEQID NO:119 D-D-Y-G-L-D-N ηV1-1 SEQ ID NO:120 N-G-Y-L-R-V-R ηV1-2 SEQ IDNO:121 E-A-V-G-L-Q-P-T ηV1-3 SEQ ID NO:122 L-A-V-F-H-E-T-P-L-G-Y ηV1-4SEQ ID NO:123 D-F-V-A-N-C-T-L ηV1-5 SEQ ID NO:124 W-V-D-L-E-P-E-G-K-VηV1-6 SEQ ID NO:125 H-S-L-F-K-K-G-H ηV1-7 SEQ ID NO:126T-G-A-S-D-T-F-E-G ηV5-1 SEQ ID NO:127 E-G-H-L-P-M ηV5-1.1 SEQ ID NO:128E-G-H-D-P-M ηV5-2 SEQ ID NO:129 I-K-S-R-E-D-V-S ηV5-3 SEQ ID NO:130V-R-S-R-E-D-V-S ηV5-4 SEQ ID NO:131 P-R-I-K-S-R-E-D-V λV1-1 SEQ IDNO:132 H-Q-V-R-V-K-A-Y-Y-R λV1-2 SEQ ID NO:133 Y-E-L-N-K-D-S-E-L-L-IλV3-1 SEQ ID NO:134 M-D-Q-S-S-M-H-S-D-H-A-Q-T- V-I λV3-2 SEQ ID NO:135L-D-Q-V-G-E-E λV3-3 SEQ ID NO:136 E-A-M-N-T-R-E-S-G λV5-1 SEQ ID NO:137D-D-I-V-R-K μV5-2 SEQ ID NO:138 V-K-L-C-D-F-G-F μV5-2.1 SEQ ID NO:139I-R-L-C-D-F-A-F μV5-3 SEQ ID NO:140 Q-V-K-L-C-D-F-G-F-A μV1-1 SEQ IDNO:141 M-S-V-P-P-L-L-R-P μV1-2 SEQ ID NO:142 K-F-P-E-C-G-F-Y-G-L-Y μV3-1SEQ ID NO:143 D-P-D-A-D-Q-E-D-S μV3-2 SEQ ID NO:144 S-K-D-T-L-R-K-R-HμV3-3 SEQ ID NO:145 I-T-L-F-Q-N-D-T-G μV3-4 SEQ ID NO:146G-S-N-S-H-K-D-I-S μV5-1 SEQ ID NO:147 S-D-S-P-E-A ΘV1-1 SEQ ID NO:148G-L-S-N-F-D-C-G ΘV1-2 SEQ ID NO:149 Y-V-E-S-E-N-G-Q-M-Y-I ΘV1-3 SEQ IDNO:150 I-V-K-G-K-N-V-D-L-I ΘV1-4 SEQ ID NO:151 D-M-N-E-F-E-T-E-G-F ΘV3-1SEQ ID NO:152 C-S-I-K-N-E-A-R-L ΘV3-2 SEQ ID NO:153 G-K-R-E-P-Q-G-I-SΘV3-3 SEQ ID NO:154 D-E-V-D-K-M-C-H-L ΘV5-1 SEQ ID NO:155 R-A-L-I-N-SΘV5-2 SEQ ID NO:156 V-K-S-P-F-D-C-S ΘV5-2.1 SEQ ID NO:157V-R-S-P-F-D-C-S ΘV5-3 SEQ ID NO:158 D-R-A-L-I-N-S ιV5-1 SEQ ID NO:159I-S-G-E-F-G-L-D ιV5-1.1 SEQ ID NO:160 C-S-G-E-F-G-L-D ιV5-2 SEQ IDNO:161 D-D-D-I-V-R-K ιV5-3 SEQ ID NO:162 D-D-I-V-R-K

As discussed more fully below, it is preferable that the PKC modulatorypeptide be chemically associated with a transport peptide. In aparticularly preferred embodiment, the modulatory peptide and thetransport peptide are linked via a disulfide bond. In the case of theforming a disulfide bond, it may be advantageous to add Cys residue tothe PKC modulatory peptide sequence, preferably at the amino terminus ofthe peptide. Alternatively, an endogenous Cys residue can be exploitedto link the modulatory peptide with the transport peptide or moiety.Methods of forming disulfide bonds are well known to those of ordinaryskill in the art, for example mixing components in a reducingenvironment and then introducing the components to an oxidizingenvironment.

Transport Peptide

A wide variety of molecules (particularly macromolecules such aspeptides) intended for cellular uptake were found to be transportedpoorly across cell membranes. Among the solutions proposed to facilitatecellular uptake have been the use of transporter moieties such ascationic (i.e., positively charged) polymers, peptides and antibodysequences, including polylysine, polyarginine, Antennapedia-derivedpeptides, HI Tat-derived peptides and the like. (See, for example, U.S.Patents and Publications U.S. Pat. Nos. 4,847,240, 5,652,122, 5,670,617,5,674,980, 5,747,641, 5,804,604, 5,888,762, 6,316,003, 6,593,292,US2003/0104622, US2003/0199677 and US2003/0206900, all of which arehereby incorporated by reference in their entirety.)

A particular example of a peptide/transporter conjugate is KAI-9803 (SEQID NO: 1), which is made up of a δPKC-derived peptide and a HIVTat-derived transporter peptide. It is currently being developed forhuman therapeutic use in the treatment of reperfusion injury. As withmost pharmaceutical active agents, KAI-9803 is prepared as apharmaceutical formulation with certain stability, tolerability andbioavailability requirements.

Excipients and Anti-Aggregants

Pharmaceutically acceptable excipients suitable for use as carriers ordiluents are well known in the art, and may be used in a variety offormulations. See, e.g., Remington's Pharmaceutical Sciences, 18thEdition, A. R. Gennaro, Editor, Mack Publishing Company (1990);Remington: The Science and Practice of Pharmacy, 20th Edition, A. R.Gennaro, Editor, Lippincott Williams & Wilkins (2000); Handbook ofPharmaceutical Excipients, 3rd Edition, A. H. Kibbe, Editor, AmericanPharmaceutical Association, and Pharmaceutical Press (2000); andHandbook of Pharmaceutical Additives, compiled by Michael and Irene Ash,Gower (1995).

Lyophilized formulations are typically prepared from an active agentdissolved in a pharmaceutically acceptable solvent, optionally includingexcipients such as bulking agents, solubility enhancers, pH buffers andthe like. The solution is subjected to reduced temperatures and pressureto drive off the liquids, leaving a solid cake that can be stored forfuture use.

The lyophilized formulations of the disclosed invention advantageouslyinclude an anti-aggregant, such as a sugar, where such sugars aresufficient to interact with the active agents' hydrophobic and/orpositively charged portions to favor their organization with the sugar,rather than aggregation with each other. Suitable anti-aggregant sugarsinclude fructose, lactose, glycerol, mannitol and D-mannose, preferablymannitol.

The transport moieties used to facilitate cellular uptake of peptides(such as the δPKC sequence portion of KAI-9803) share certain attributes(generally being cationic) that contribute to their functionality invivo, but have been discovered to give rise to the formation ofaggregates under lyophilized storage conditions. The modulatory peptidesmay also possess structural features which facilitate the formation ofaggregates. While not wishing to be bound to any particular theory, suchaggregation may result from peptide dimerization and a tendency for thepeptides to “organize” into aggregates.

The formation of a detrimental level of aggregates interferes withre-dissolution of peptides and peptide conjugates, in turn interferingwith administration where the possibility of particulates would beunacceptable for certain routes of administration (e.g., intracoronary).Notwithstanding these drawbacks, the creation of a detrimental level ofaggregates in the formulation complicates determining finalconcentration in that the precise amount of peptide dissolved per unitof liquid. Such a determination cannot be accurately calculated withoutfirst determining and then subtracting the weight of undissolvedmaterial. Unacceptable levels of aggregation can result in from 0.1 to50% aggregation of the peptide conjugates. Thus, another aspect of thepresent invention pertains to the incorporation of an anti-aggregant inlyophilized formulations of peptides or peptide/transporter conjugates.In addition to suppressing the formation of aggregants in thelyophilized product, certain anti-aggregants can enhance thestabilization of the pharmaceutical formulation.

Administration

Parenteral administration is generally characterized by injection,either subcutaneously, intramuscularly, intraperitoneal, intravenously,and in the case of the present invention via intracoronary injection.Injectables can be prepared in conventional forms, either as liquidsolutions or suspensions, solid (e.g., dried or lyophilized) formssuitable for reconstitution into solution or suspension in liquid priorto injection, or as emulsions. Generally, suitable excipients include,for example, water, saline, dextrose, glycerol, ethanol or the like. Inaddition, minor amounts of non-toxic auxiliary substances can beemployed, such as wetting or emulsifying agents, pH buffering agents,solubility enhancers, tonicifiers and the like including, for example,sodium acetate, sorbitan monolaurate, triethanolamine oleate,cyclodextrins, etc. Dosage forms for intravenous (IV) administrationgenerally comprise an active agent incorporated into a sterile solutionof simple chemicals such as sugars, amino acids or electrolytes, whichcan be easily carried by the circulatory system and assimilated. Suchsolutions are typically prepared with saline or buffer. The pH of suchIV fluids may vary, and will typically be from 3.5 to 8.0, as known inthe art.

The intracoronary injection formulations of the disclosed invention aretypically prepared using sodium chloride for injection, USP (preferably0.9%) for reconstitution of the lyophilized API into solution atconcentrations ranging from 0.001 to 2.5 mg/mL, preferably 0.01 to 1.0mg/mL.

Exemplary Formulations

The pharmaceutical formulations of the disclosed invention preferablyinclude a PKC modulatory peptide, a transport peptide, and ananti-aggregant agent. Typically, the PKC modulatory peptide and thetransport peptide are chemically associated with one another. Forexample, it is preferred that the modulatory peptide and the transportpeptide are covalently bonded to one another. In a preferred embodiment,the PKC modulatory peptide and the transport peptide are linked via adisulfide bond.

In a pre-lyophilized embodiment of the invention, the formulationfurther includes a sufficient amount of a pharmaceutically acceptablesolvent (preferably water for injection, USP) to solubilize theforegoing components. A lyophilized embodiment of the invention includescomponents described above in the form of a solid cake.

The ratio of peptides to anti-aggregant in the disclosed formulationsranges from about 100:1 to about 5:1, preferably from about 80:1 toabout 5:1, more preferably from about 80:1 to about 8:1; the actualratio will depend upon the identity of the components and theconcentration desired for the lyophilized and/or reconstituted drugproducts.

One aspect of the present invention provides a pre-lyophilizedformulation for a peptide or peptide/transporter conjugate, as follows:

TABLE 2 Ingredient Amount (wt/vol) API 0.25 to 5.0 mg/mL Anti-aggregantSugar 2.0 to 50.0 mg/mL WFI (USP) q.s. to 100and the lyophilized product therefrom.

Another aspect of the preferred peptide or peptide/transporter conjugateformulation can be obtained by lyophilization of the following:

TABLE 3 Ingredient Amount (wt/vol) API 0.1 to 10.0 mg/mL Anti-aggregantSugar 5.0 to 40.0 mg/mL WFI (USP) q.s. to 100

A preferred peptide or peptide/transporter conjugate formulation can beobtained by lyophilization of the following:

TABLE 4 Ingredient Amount (wt/vol) API  2.0 to 3.0 mg/mL Anti-aggregantSugar 15.0 to 25.0 mg/mL WFI (USP) q.s. to 100

Another preferred peptide or peptide/transporter conjugate formulationcan be obtained by lyophilization of the following:

TABLE 5 Ingredient Amount (wt/vol) API  0.5 to 5.0 mg/mL Anti-aggregantSugar 10.0 to 30.0 mg/mL WFI (USP) q.s. to 100

Still another preferred peptide or peptide/transporter conjugateformulation can be obtained by lyophilization of the following:

TABLE 6 Ingredient Amount (wt/vol %) API  2.0 to 3.0 mg/mLAnti-aggregant Sugar 15.0 to 25.0 mg/mL WFI (USP) q.s. to 100

A further preferred peptide/transporter conjugate formulation can beobtained by lyophilization of the following:

TABLE 7 Ingredient Amount KAI-9803 5.0 mg Mannitol (USP) 40.0 mg WFI(USP) 2.0 mL

Alternatively, aqueous parenteral solutions of KAI-9803 can be preparedsubstantially free of sugars, at concentrations ranging from about 0.01to about 10.0 mg/mL, preferably about 0.1 to about 5.0 mg/mL, and mostpreferably about 0.1 to about 1.0 mg/mL. The pH of such aqueoussolutions is adjusted to between about 2.0 and 4.0, preferably betweenabout 2.5 and 3.5.

Methods of Manufacture and Use

The PKC modulatory peptides and the transporter peptides can besynthesized according to conventional (e.g., solid phase) procedures.After activation of the Cys on one of the PKC modulatory and transporterpeptides [e.g., using 2,2′-dithiobis(5-nitropyridine) to activate thecarrier peptide] the two peptides are coupled, isolated and thenpurified [e.g., by preparative RP-HPLC using acetonitrile elution in aTEAP buffer (triethylamine and phosphoric acid) giving rise to thepurified phosphate salt]. The fractions from the HPLC containing thepurified and coupled peptides are then pooled. A pharmaceuticallyacceptable salt can be exchanged by repeat RP-HPLC eluting withacetonitrile and the desired organic or inorganic acid counter-ion donor(such as acetic acid, hydrochloric acid, tartaric acid and the like,preferably acetic acid). The desired end product is pooled, divided intolyophilization flasks, lyophilized, and transferred to suitablecontainers for storage prior to formulation (preferably in sealed amberglass containers at reduced temperature, e.g., −20° C.).

The counterion employed during the production of KAI-9803 has a positiveeffect on solubility and stability. An acetate counterion is used in thepreferred embodiment. Other counterions, such as a chloride counterionare also contemplated. And while use of an acetate counterion is apreferred embodiment of the disclosed invention, it is not required.

The pharmaceutical formulations of the present invention can bemanufactured according to most accepted practices, for example, asfollows:

-   (a) Appropriate amounts of anti-aggregant, hydrophobic active agent    and/or cationic transport moiety are introduced to a suitably sized    container (preferably a glass vial) as dry solids.-   (B) A pharmaceutically acceptable solvent [e.g., water for injection    (“WFI”)] is added to the container in an amount sufficient to    dissolve the solids and attain a desired concentration.-   (C) The solution thus-formed is filtered, aseptically dispensed into    a pre-sterilized container, and lyophilized to dryness.-   (D) The container is sealed (optionally after first filling the    head-space with a non-reactive gas, such as nitrogen) for storage    until reconstitution for administration.

It is recommended that such pharmaceutical products be stored at orbelow room temperature, preferably at about 2-8° C. (more preferably 5°C.), which instructions should be displayed on the container or itsattached label, its outer packaging and any package insert includedtherein.

To reconstitute the lyophilized formulation for administration, theproduct is first warm to about room temperature before opening. Shortlyprior to use, the sealed container is accessed via a needle through therubber stopper and a pharmaceutically acceptable solvent (such assaline, preferably 9% saline) is added in an amount sufficient tosolubilize the lyophilized cake and provide the desired concentrationfor administration. Such instructions for reconstitution can be providedin a pharmacy manual, on dosing cards, or can be incorporated on thecontainer or its attached label, outer packaging and/or package insert.

Testing

Testing of the pharmaceutical formulations of the present invention canbe accomplished by procedures well known in the art, for example,including: determination of active pharmaceutical ingredient identityand concentration by HPLC-UV (measuring absorbance at 206, 220 and/or280 nm) e.g., before and after lyophilization and reconstitution;determination of water content in lyophilized product; pH ofpre-lyophilized solution and reconstituted solution; and appearance oflyophilized cake.

EXAMPLES

The following examples serve to describe more fully the manner of usingthe above-described invention, as well as to set forth the best modescontemplated for carrying out various aspects of the invention. It isunderstood that these examples in no way serve to limit the true scopeof this invention, but rather are presented for illustrative purposes.All references cited herein are incorporated by reference in theirentirety.

Example 1 Manufacture of KAI-9803 Acetate

A. Peptide Fragment Synthesis

Merrifield resin is pre-swelled in dichloromethane (DCM) for at least 2hours. The DCM is drained. Transporter and δ-PKC peptides are preparedby solid phase synthesis as follows:

-   1. deprotection (TFA/DCM),-   2. resin washing (2-propanol, methanol, 10% TEA/DCM, methanol and    DCM),-   3. coupling of the next amino acid residue (t-Boc-AA-OH, using    HOBt/HBTU/NMM), and-   4. resin washing (methanol and DCM)].

Deprotection and coupling are monitored by performance of a ninhydrintest. After incorporation of the final amino acid residue on eachpeptide (Cys), the resin peptide is deprotected and washed (steps 1, 2and 4, above). The peptide-resin bond and side chain protecting groupsare cleaved by treatment with HF/anisole and precipitated by ethylether.

B. Peptide Fragment Purification and Isolation

Transporter and δ-PKC peptides obtained, e.g., as described in ExampleIA are subjected to preparative RP-HPLC on a C-18 column, using anacetonitrile gradient in trifluoroacetic acid solution. The acceptancecriterion for purity of these intermediate peptides is not less than90.0%.

C. Coupling

Transporter peptide obtained, e.g., as described in Example IB isactivated by contact with 2,2′-dithiobis(5-nitropyridine) and thencontacted with the δ-PKC peptide from Example IB to afford the coupledpeptide conjugate KAI-9803.

D. Purification, Salt Exchange and Isolation

Crude KAI-9803 obtained, e.g., as described in Example IC is purified bypreparative RP-HPLC using an acetonitrile elution in a TEAP buffer(triethylamine and phosphoric acid) on a YMC C-18 column. The fractionsresulting from the purification are analyzed by an analytical RP-HPLCin-process method. Those fractions that meet the purity criterion (notless than 95%) are pooled, loaded back onto the same C-18 column andeluted with acetonitrile in an acetic acid buffer to give thecorresponding KAI-9803 acetate salt. The thus-purified KAI-9803 acetatesalt is pooled, divided into lyophilization flasks and frozen. Thefrozen flasks are connected to a lyophilizer manifold and thelyophilization is performed. Upon completion of lyophilization, theresulting KAI-9803 acetate powder is weighed, samples are taken fortesting, and the remainder transferred into 50 mL amber glasscontainers. The containers are closed with 20 mm, (grey)butyl, snap-onstoppers, and stored at −20° C.

Example 2 Formulation, Lyophilization, Fill and Finish

KAI-9803 acetate powder (50.0 mg) obtained, e.g., as described inExample ID and mannitol USP (400.0 mg) are dissolved in about 14.0 mL ofWFI, followed by the addition of WFI as necessary to total 20 ml (˜6 ml)to give a clear, colorless solution. Clarity, color and completedissolution of solids are confirmed by visual examination. The solutionis aseptically filtered through two serial 0.22 μm filters into a class100 aseptic filling suite. Two mL of the filtered solution areaseptically dispensed into each of ten pre-sterilized 20 mL vials. Eachvial is capped with a slotted lyophilization stopper and loaded ontoshelves pre-chilled to −50° C. A primary drying cycle is performed at ashelf temperature of 5° C. for not less than 20 hours, followed by asecondary drying step with a shelf temperature of 25° C. for not lessthan 3 hours. Upon completion of the lyophilization cycle, the vials arestoppered under nitrogen with a partial vacuum and sealed. The stopperedvials are crimped and inspected in a class 10,000 processing suite. Thevials are labeled and then moved to 2-8° C. storage under quarantine.

Example 3 Reconstitution of a KAI-9803+Mannitol Formulation

A vial containing a lyophilized pharmaceutical formulation of 5 mgKAI-9803 and 40 mg mannitol (obtained, e.g., as described in Examples 1and 2) is injected with 20 mL of 0.9% sodium chloride for injection,USP, is added to the vial and the contents are dissolved with gentleswirling to yield a clear solution. To a sterile, empty IV bag is added18 mL of 0.9% sodium chloride for injection, USP, followed by theaddition of 2 mL of KAI-9803 solution (taken from the vial) to yield atotal volume of 20 mL of a 0.1 mg/mL solution of KAI-9803 in the IV bag.The solution is stored at room temperature and used within 4 hours ofpreparation.

Example 4 Stability of Lyophilized KAI-9803 Formulations

Formulations of KAI-9803 are prepared, for example as described inExamples 1 and 2, using mannitol and substituting mannitol with fructoseand sucrose as the anti-aggregant sugar for the formulating procedure ofExample 2. All of the solutions are visually inspected for clarity,color and complete dissolution of solids, and an aliquot is removed fromeach. Each aliquot is analyzed for KAI-9803 concentration using HPLC-UV,measuring absorbance at 206 and 280 nm. The remaining solutions arefiltered, filled, lyophilized and finished, e.g., as described inExample 2. One set of vials representing each of the formulations isseparated for immediate reconstitution and testing (HPLC-UV @ 206/280nm) to confirm KAI-9803 concentration in the reconstituted product. Theremaining vials are divided into groups for storage at reducedtemperature (e.g., 2-8° C.), at room temperature, and at elevatedtemperature (e.g., 35° C.). Sets of vials representing each of theformulations are withdrawn at selected time points (e.g., 1 day, 1 week,1 month, 3 months, 6 months), are reconstituted, visually inspected andtested for KAI-9803 concentration (HPLC-UV @ 206/280 nm).

While the present invention has been described with reference to thespecific embodiments thereof, it should be understood by those skilledin the art that various changes may be made and equivalents may besubstituted without departing from the true spirit and scope of theinvention. In addition, many modifications may be made to adapt aparticular situation, material, composition of matter, process, processstep or steps, to the objective, spirit and scope of the presentinvention. All such modifications are intended to be within the scope ofthe claims appended hereto. All patents and publications cited above arehereby incorporated by reference.

1. A pharmaceutical formulation, comprising: a protein kinase Cmodulatory peptide chemically coupled to a transport peptide, whereinthe modulatory peptide coupled to the transport peptide comprising theamino acid sequence of SEQ ID NO:1; and a sugar serving as an excipient,wherein the sugar and the modulatory peptide coupled to the transportpeptide are in a ratio from 100:1 to about 1:1.
 2. The formulation ofclaim 1, wherein the sugar is selected from the group consisting offructose, lactose, D-mannose and mannitol.
 3. The formulation of claim1, wherein the ratio is from about 80:1 to about 8:1.
 4. The formulationof claim 1, wherein the ratio is from about 80:1 to about 5:1.
 5. Theformulation of claim 1, wherein the formulation is lyophilized.
 6. Theformulation of claim 1, wherein the ratio is about 5:1.
 7. Theformulation of claim 1, wherein the formulation is suitable for aparenteral route of administration.
 8. The formulation of claim 7,wherein the parenteral route of administration is intracoronaryadministration.